Summary

LIM homeobox family members regulate a variety of cell fate choices during
animal development. In C. elegans, mutations in the LIM homeobox gene
lin-11 have previously been shown to alter the cell division pattern
of a subset of the 2° lineage vulval cells. We demonstrate multiple
functions of lin-11 during vulval development. We examined the fate
of vulval cells in lin-11 mutant animals using five cellular markers
and found that lin-11 is necessary for the patterning of both 1°
and 2° lineage cells. In the absence of lin-11 function, vulval
cells fail to acquire correct identity and inappropriately fuse with each
other. The expression pattern of lin-11 reveals dynamic changes
during development. Using a temporally controlled overexpression system, we
show that lin-11 is initially required in vulval cells for
establishing the correct invagination pattern. This process involves
asymmetric expression of lin-11 in the 2° lineage cells. Using a
conditional RNAi approach, we show that lin-11 regulates vulval
morphogenesis. Finally, we show that LDB-1, a NLI/Ldb1/CLIM2 family member,
interacts physically with LIN-11, and is necessary for vulval morphogenesis.
Together, these findings demonstrate that temporal regulation of
lin-11 is crucial for the wild-type vulval patterning.

INTRODUCTION

Specification of different cell types during development involves multiple
cell-cell interactions mediated by many genes. Studies of the C.
elegans hermaphrodite vulva have proven successful in dissecting
regulatory networks and understanding the function of some crucial genes. The
adult vulva is formed by the progeny of three out of six vulval precursor
cells (VPCs) that acquire 1° and 2° fates in a 2°-1°-2°
pattern, and undergo three rounds of cell divisions
(Sulston and Horvitz, 1977)
(Fig. 1). During L4 stage,
vulval cells differentiate into seven different cell types and form an
invaginated structure (Sharma-Kishore et
al., 1999) (Fig.
1). Vulval development thus provides opportunities to study cell
fate specification and pattern formation. Mutations that perturb vulval
morphology have identified some of the genes that regulate cell
differentiation, including lin-17 (frizzled family),
lin-18 and lin-11 (LIM homeobox family)
(Ferguson and Horvitz, 1985;
Ferguson et al., 1987;
Freyd et al., 1990;
Sawa et al., 1996;
Gupta and Sternberg, 2002).
Mutations in these genes alter the axes of terminal cell division, suggesting
a role in differentiation of a subset of the vulval cells. lin-11 is
known to be necessary for the NT portion of the 2° lineage vulval cells
because there is a LLLL cell lineage pattern in lin-11 mutant animals
compared with the wild-type NTLL pattern (see
Fig. 1)
(Ferguson et al., 1987). Being
a LIM homeodomain family member, LIN-11 is likely to function as a
transcriptional regulator of vulval cell-type specific genes.

Wild-type vulval development. During L2/L3 stages, the anchor cell (AC)
induces P5.p, P6.p and P7.p VPCs to adopt 1° and 2° cell fates. The
relative positions of the cell nuclei have been drawn. The terminal cell
division axes are NTLL for the 2° lineage and TTTT for the 1° lineage
(N, not divided; T, transverse; L, longitudinal). The Pn.pxxx cells invaginate
during L4 stage to give rise to the future vulval opening. The seven
differentiated cell types have been marked with colors (1° lineage vulE
and vulF are red; 2° lineage vulA are white; vulB1 and vulB2 are yellow;
vulC are light green; and vulD are dark green).

The C. elegans genome encodes seven LIM homeodomain proteins
including LIN-11 (Ruvkun and Hobert,
1998). LIN-11 has been shown to be necessary for the development
of a subset of vulval cells, uterine π lineage cells and some neurons
(Ferguson et al., 1987;
Hobert et al., 1998;
Newman et al., 1999;
Sarafi-Reinach et al., 2001;
Gupta and Sternberg, 2002). In
this study, we show that the spatiotemporal expression of lin-11
confers distinct cell fates. Our experiments reveal at least two distinct
functions of lin-11 in vulval cells. lin-11 is first
required for setting up the correct pattern of vulval invagination. During
this phase, the precursors of vulC and vulD express high levels of
lin-11. Later on, lin-11 is expressed in all vulval progeny.
Using a conditional RNAi approach, we have examined lin-11 function
during vulval morphogenesis and demonstrate that lin-11 is required
in vulval progeny for wild-type patterning. Finally, we show that the
LIM-binding protein LDB-1 (Cassata et al.,
2000) plays a role in vulval differentiation by directly
interacting with LIN-11.

MATERIALS AND METHODS

Strains and culture conditions

C. elegans were grown and mutagenized according to published
methods (Brenner, 1974;
Wood, 1988). Except for the
heat shock strains, for which a 15°C incubator was used, all other strains
were maintained at 20°C. Mutations and transgenes used are as follows.

Laser ablations were performed as described by Avery and Horvitz
(Avery and Horvitz, 1987).
Early- to mid-L3 stage worms were chosen for the study.

Heat-shock experiments

hs::lin-11 animals (syEx500 and syEx530) were
pulsed at different temperatures (between 30°C to 33.5°C) and duration
(15 minutes to 1 hour) during Pn.px and Pn.pxx stages. In general, stronger
pulses (1 hour at 31°C or 30 minutes at 33.5°C) caused growth arrest,
uncoordinated movement and larval lethality. These are probably the result of
interference with the function of other LIM homeobox genes
(Ruvkun and Hobert, 1998).
Alternatively, high levels of lin-11 expression in neurons may
interfere with their normal development
(Hobert et al., 1998;
Sarafi-Reinach et al., 2001).
For the vulval phenotypes, we used a 20 minutes heat shock at 33°C.

lin-11 RNAi animals (hs-dslin-11i) were heat shocked
during early Pn.pxxx stage for 1 hour at 33°C. After recovery at 20°C,
vulval phenotypes were examined during mid-L4 stage.

Molecular biology

hs::lin-11 construct (pPHS11.82)

To construct pPHS11.82, a 1.5 kb NsiI-KpnI
lin-11 genomic fragment from the cosmid ZC247 was cloned into
pPD49.83 (Mello and Fire,
1995). As the construction deleted 125 bp of the hsp16-41
promoter, it was restored as a NsiI fragment by PCR amplification of
the vector DNA using primers FBG3 (5′CGGCTCGTATGTT-GTGTGGAATTG3′)
and BBG2 (5′CGCGATGCATGATGAGG-ATTTTCGAAGTTTTTTAG3′). The resulting
construct was digested with SphI and KpnI to obtain a 1.9 kb
DNA fragment. In a separate experiment, a 10.8 kb ZC247 NcoI fragment
was inserted in pPD49.83 to obtain pPHS11.108. pPHS11.108 was digested with
SphI and KpnI and subsequently ligated with the 1.9 kb
SphI-KpnI fragment to obtain pPHS11.82. The beginning and
end sequences (18 nucleotides) of the lin-11 genomic fragment are
5′-ATGCATTCTTCTTCTTCG-3′ and
5′-CCATGGTTCCTATGAGGT-3′.

ldb-1::GFP construct (pLBP13.3c)

The cosmid F58A3 was digested with SphI and PstI and a
13.3 kb fragment was subcloned into pPD95.73 (a gift of A. Fire, S. Xu, J.
Ahnn and G. Seydoux). The beginning and end sequences (18 nucleotides) of the
fragment are: 5′-GCATGC-TTTTTTTTAATT-3′ and
5′-CTGCAGCTGTAGCTTTTT-3′.

ldb-1 RNAi construct (pYK66F4-1)

ldb-1 cDNA (yk66f4, kindly provided by Dr Yuji Kohara, National
Institute of Genetics) was digested with EcoRI and NotI and
a 720 bp fragment was subcloned in pBS-SK(+). In vitro RNA was synthesized
using the Ambion MEGAscript kit.

ldb-1 RNAi experiments

ldb-1 RNAi was performed by soaking
(Tabara et al., 1998). An
equal amount of each RNA strand (20 μl) was mixed to generate dsRNA. For
control RNA, the pBS-SK(+) vector with no ldb-1 insert was used.
Worms were synchronized by 24 hours L1 starvation in M9 after bleach treatment
of the adult hermaphrodites. A small aliquot of the L1 stage worms was mixed
with dsRNA solution (5 to 20 μl) and 1-5 μl OP50 bacteria. Worms were
incubated for 30-36 hours, at which time they were washed twice with M9 and
transferred to regular plates seeded with OP50. L4 stage animals were examined
for the vulval phenotype and GFP expression.

Two-hybrid experiments

Two-hybrid experiments were performed as suggested by the manufacturer
(Clontech/BD Biosciences). pGBKT7 (GAL4 DNA binding) and pGADT7
(GAL4 activation) vectors were used to subclone lin-11 and
ldb-1 cDNA fragments, respectively. Positive control vectors were
pVA3 (murine p53 insert) and pTD1 (SV40 large T-antigen insert). To subclone
lin-11 LIM domains, a 682 bp product was PCR amplified using primers
lin-11-LIM-u1 (5′GGCATATGACCTCACTGGAAGAAGAGGAG3′) and
lin-11-LIM-d1 (5′GGGTCGACTCGAGTCATCTGAATTGTCCTTC3′) and
lin-11 cDNA as a template. The resulting product was digested with
NdeI and SalI and subcloned in pGBKT7. ldb-1 LID
region (553 bp) was PCR amplified using primers ldb-1-LID-u1
(5′GGCA-TATGGGAAGCAAAAAAGCTACAGCTG3′) and ldb-1-LID-d1
(5′GGCTCGAGGTGGCATCCGACTATTCGGCATC3′) and the template
ldb-1 cDNA. PCR product was digested with NdeI and
XhoI and subcloned in pGADT7. Transformed AH109 yeast cells were
grown on SD/–Leu/–Trp and SD/–His/–Leu/–Trp
plates at 30°C.

RESULTS

lin-11 mutant vulval cells exhibit abnormal cell
fusions

Previous studies of lin-11 function during vulval development have
defined its requirements during differentiation of a subset of the vulval
progeny: vulC and vulD (Ferguson et al.,
1987). Two experiments suggested that lin-11 might be
involved in patterning additional vulval cells. First, in a screen for mutants
with altered patterns of egl-17::GFP and ceh-2::GFP
expression, three lin-11 alleles (sy533, sy534 and
sy634) were recovered. Second, ablation of the daughters of P5.p and
P7.p VPCs in lin-11 mutant animals did not alter the invagination
defect of P6.p progeny, suggesting that the effects on 1° lineage are not
an indirect consequence of the 2° lineage defect. To analyze the defects
in lin-11 mutant vulval cells further, we used the
ajm-1::GFP cell-junction marker (formerly known as MH27::GFP
and jam-1::GFP) that reveals cell boundaries and allows the in vivo
study of cell fusion events (Podbilewicz
and White, 1994; Mohler et
al., 1998; Raich et al.,
1999). Sharma-Kishore et al. have used the marker to describe the
process of wild-type vulval development
(Sharma-Kishore et al.,
1999).

We examined ajm-1::GFP vulval expression in mid-L4 stage animals.
Fig. 2A shows a typical
wild-type expression (ventral view) in the form of seven concentric rings that
arise from specific fusion between vulval cells
(Sharma-Kishore et al., 1999).
These rings are visible at different focal planes since vulval cells have
invaginated significantly (see Fig.
2B for a schematic representation). Each of the vulval rings
represents one cell type (vulA-vulF; Fig.
2B). By contrast, ajm-1::GFP expression in
lin-11(n389) animals reveals dramatic defects in cell fusion events.
In all 12 animals examined, only two or three vulval rings could be seen;
moreover, the rings were visible in the same focal plane (Fig.
2C,D,F,G).
One of these rings was unusually large (big arrowheads in Fig.
2C,D)
and encircled one or two smaller odd-shaped rings (small arrows in Fig.
2C,D).
The big ring corresponds to the P5.p and P7.p progeny that have fused
together, while the smaller ones belong to the P6.p progeny. In 30% of these
animals some of the 2° lineage cells did not fuse with the large syncytium
and remained isolated (red star brackets in Fig.
2C,F).
Some of these cells (two to six) showed punctate ajm-1::GFP
expression, suggesting partial fusion with the surrounding hyp7 syncytium
(Fig. 2C). To confirm that the
large syncytium is 2° lineage specific, we ablated the P6.p progeny during
early L3 stage and found no change in the formation of large syncytium
(n=4; Fig.2E).

Cell fusion events in vulva revealed by the expression of
ajm-1::GFP. (A,B) Wild-type. (C-G) lin-11(n389). The regions
occupied by VPC progeny are marked with brackets. All images are the ventral
views of the vulva. (A) Wild-type vulval rings (vulA to vulF) are visible at
different focal planes. The outermost boundaries of the rings are marked with
respective cell types. (B) A schematic representation of the vulval rings from
lateral and ventral sides. For easier viewing, alternative rings have been
coded with dark and light colors. The ventral view shows all seven rings
together. (C) A n389 animal with abnormal vulval fusions. The
outermost ring (big arrow) corresponds to the 2° lineage cells. The inside
rings (small arrows) belong to the 1° lineage cells. Brackets marked with
red star point to the unfused vulval cells (punctate GFP). (F) Same animal as
in C, under Nomarski. (D) Another n389 animal. In this case, all
2° lineage cells have fused together (big arrow). The small arrows point
to the 1° lineage rings. (G) Same animal as in D, under Nomarski. (E) P6.p
lineage cells were ablated in this n389 animal. The outermost big
ring is still visible (arrow). (H) n389 animal. P5.p and P7.p lineage
cells were ablated during early L3 stage. Vulval rings corresponding to the
1° lineage cells (arrows) have abnormal morphology. Scale bars: in A, 12μ
m; in G, 10 μm for C-H.

We also observed defects in ajm-1::GFP expression in the 1°
lineage vulval cells. In these cases, the defect appeared to be the smaller
size of the vulE and vulF rings, and their failure to align correctly (small
arrows in Fig.
2C,D).
To determine whether the 1° lineage defect is due to the autonomous
requirement of lin-11, we ablated P5.p and P7.p cells during early L3
stage. Such ablated animals still showed morphological defects in vulE and
vulF rings (n=3, Fig.
2H). Together, these results provide strong evidence that
lin-11 is necessary for the development of all 1° and 2°
lineage vulval cells. We did not observe any cell fusion defect in
lin-11(n389) animals at the earlier stages of vulval development.

egl-17 expression in vulval cells has been shown to be a faithful
marker of wild-type development (Burdine et
al., 1998; Ambros,
1999; Wang and Sternberg,
1999). The earliest expression of egl-17::GFP is detected
in P6.p. The expression continues in the P6.p lineage during Pn.px (VPC
progeny after first cell division, x denotes both anterior and posterior
cells) and Pn.pxx (VPC progeny after second cell division) stages. However,
after third cell division of VPCs (Pn.pxxx cells) egl-17::GFP
expression is no longer detected in the P6.p lineage but instead is expressed
in the vulC and vulD progeny of the 2° lineages
(Burdine et al., 1998) (Fig.
3A,B).
We find that early expression of egl-17::GFP in P6.p lineage is not
altered in lin-11(n389) animals. However, during the Pn.pxxx stage,
the 2° lineage cells fail to express a detectable level of GFP
(Burdine et al., 1998) (Fig.
3C,D;
Table 1). However, the P6.p
lineage cells continue to express low levels of GFP, suggesting a
defect in their differentiation (Fig.
3C,D).
Thus, vulC, vulD cells (2° lineage) and vulE, vulF cells (1° lineage)
have not acquired the correct identity.

Expression pattern of the vulval markers in wild-type and
lin-11(n389) vulva. (A,C,E,G,I,K) Nomarski images of the L4 stage
vulval cells. (B,D,F,H,J,L) Corresponding images showing GFP fluorescence.
Primary lineage cells have been marked with stars and 2° lineage cells
with arrows. Anterior is towards the left. (A,B) Wild-type
egl-17::GFP expression in the vulC and vulD cells. (C,D) In
n389 background presumptive vulC and vulD do not reveal any
egl-17::GFP. Instead, weak egl-17::GFP is expressed in all
the 1° lineage cells. (E,F) Wild-type cdh-3::GFP expression is
detected in all the 1° lineage cells and in the vulC, vulD of the 2°
lineage cells. (G,H) n389; cdh-3::GFP animals have no
detectable GFP fluorescence in the 2° lineage cells. In this animal, the
presumptive vulF cells are expressing weak GFP, whereas vulE reveal
no detectable expression. (I,J) Wild-type zmp-1::GFP expression is
seen in the vulA and vulD of the 2° lineage. By contrast, n389;
zmp-1::GFP animals (K,L) do not express GFP in any of the
vulval cells. The bright fluorescence is seen in some uterine lineage cells.
Scale bar: 8 μm.

Cell-autonomous expression of vulval markers in wild-type and
lin-11(n389) animals

cdh-3 belongs to a cadherin superfamily of genes, members of which
are known to play various roles in epithelial morphogenesis such as cellular
adhesion and cell shape changes (Gumbiner,
1996; Pettitt et al.,
1996; Costa et al.,
1998; Hill et al.,
2001). The wild-type cdh-3::GFP (syIs50)
expression in vulval cells is detected during L4 stage in the vulC, vulD, vulE
and vulF cells (Fig.
3E,F;
Table 1). In
lin-11(n389) animals, cdh-3::GFP expression is completely
abolished in the presumptive vulC and vulD cells (Fig.
3G,H;
Table 1). In addition,
expression in the 1° lineage cells is considerably affected: presumptive
vulF shows very weak GFP fluorescence, whereas vulE shows fluorescence only on
rare occasions (Fig.
3G,H;
Table 1). Thus, lin-11
is required for the differentiation of the vulC, vulD, vulE and vulF cell
types.

The zmp-1 gene encodes a zinc metalloprotease and has been used as
a marker for the 1° lineage cells
(Wang and Sternberg, 2000;
Inoue et al., 2002).
zmp-1::GFP (syIs49) expression in the vulva begins during
the late-L4 stage, first detected in the vulD and vulE and later on in the
vulA as well (Fig.
3I,J;
Table 1)
(Wang and Sternberg, 2000;
Inoue et al., 2002). We did
not find any zmp-1::GFP expression in lin-11(n389) vulval
cells (Fig.
3K,L;
Table 1). Thus, lin-11
is also necessary for the development of the vulA cell type.

We also examined the expression of a homeodomain family member,
ceh-2, which is expressed during L4 stage in the vulB1, vulB2 and
vulC cells (syIs54) (Inoue et
al., 2002) (Table
1). In lin-11(n389) animals, vulval cells failed to
express ceh-2::GFP in any of the cell types
(Table 1).

The defects in vulval markers expression and cell fusion in lin-11
mutant animals reveal requirements of lin-11 in the specification of
both 1° and 2° lineage cells. It is possible that lin-11
functions non-autonomously to specify some of the cell fates. To address this
possibility, we carried out cell ablation experiments in wild-type and
lin-11(n389) animals. We ablated a subset of the vulval cells during
Pn.px and Pn.pxx stages, and examined expression of four GFP markers
(egl-17::GFP, zmp-1::GFP, cdh-3::GFP and ceh-2::GFP) in the
progeny of the remaining cells during L4 stage
(Table 1). Four different
ablation sets were analyzed [Set 1 (vulA, B1 and B2), Set 2 (vulC and D), Set
3 (vulA, B1, B2, C and D) and Set 4 (VulE and F)]. We found that in wild-type
control animals, all four GFP markers are expressed in cell-autonomous manner,
i.e. ablation of a subset of the vulval cells did not alter GFP
expression pattern in the progeny of the remaining cells (compare intact and
cell ablated animals in Table
1). A similar conclusion for the zmp-1::GFP was drawn
earlier by Wang and Sternberg (Wang and
Sternberg, 2000). Having examined the autonomy of the GFP markers
in wild-type animals, we carried out similar sets of cell ablations in
lin-11(n389) animals. The results, summarized in
Table 1, demonstrate that
lin-11 functions in all vulval cells and specifies their fate in
cell-autonomous manner. We can not rule out the possibility of complex
interactions. The cell fusion defects are likely to be a secondary consequence
of defects in cell fate specification.

lin-11 is dynamically expressed during vulval
development

Our analyses have revealed broader requirements for lin-11 during
vulval development. The vulval expression of lin-11 using
lin-11::lacZ reporter assays was previously reported to be in the N
and T cells of the 2° lineages (precursors of vulC and vulD) (G. A. Freyd,
PhD thesis, Massachusetts Institute of Technology, 1991)
(Struhl et al., 1993). This
pattern of expression did not provide a suitable explanation for our
observations on the lin-11 mutant phenotypes. To determine the
spatial and temporal pattern of lin-11 vulval expression precisely,
we generated several lin-11::GFP transgenic lines
(Gupta and Sternberg, 2002).
Two of these, syIs80 and syIs53, were chosen for detailed
analysis. Another lin-11::GFP integrant, nIs96
(Reddien et al., 2001), was
also analyzed. The developmental profile of the lin-11::GFP vulval
expression in all three lines is nearly identical, although their fluorescence
brightness can be ranked nIs96>syIs80>syIs53. The
syIs80 and syIs53 animals reveal dynamic changes in the
vulval GFP expression.

The earliest GFP expression in syIs80 vulval cells is
detected in one of the two daughters of the 2° lineage precursors (P5.pp
and P7.pa cells) (Fig.
4A,B,
Fig. 5A). In most cases, GFP
fluorescence was detectable only ∼1-2 hours before the VPC daughters were
beginning to divide. At this stage, expression in P6.p daughters is much
weaker and rarely observed (Fig.
5A). During the Pn.pxx stage, vulval cells begin to reveal
brighter GFP fluorescence in both the 1° and 2° lineages (Fig.
4C,D,
Fig. 5B). In the 2°
lineage, expression is typically seen in only the N and T cells
(Fig. 5B). By the Pn.pxxx
stage, lin-11::GFP expression is detected in all 2° lineage
progeny (Fig.
4E,F,
Fig. 5C). In general, vulA has
the lowest level of expression compared with others. syIs53 animals
reveal a similar pattern of expression, although the overall fluorescence is
considerably reduced (compare Fig.
5D with
5B, and Fig.
5E with
5C). The expression of
lin-11 in vulval cells is consistent with the cell fusion defects and
marker gene expression studies in lin-11 mutant animals. Together,
these results further support the hypothesis that lin-11 plays a role
in the development of all vulval cell types.

Developmental expression of lin-11::GFP (syIs80) in vulval cells.
(A,C,E) Nomarski photographs. (B,D,F) lin-11::GFP-expressing vulval
cells marked with arrows. Few VC neurons are also visible (star). (A,B) During
Pn.px stage, weak GFP fluorescence is detected in the P5.pp and P7.pa cells
(arrows in B). (C,D) By Pn.pxx stage, vulval cells express high levels of
GFP. The VPC lineage tree has been drawn. In this animal P5.ppx,
P6.ppx and P7.pax cells are seen expressing GFP. (E,F) During L4
stage, Pn.pxxx cells express GFP in the P5.p and P7.p progeny. The
region occupied by each VPC progeny has been marked. In this focal plane, only
a subset of the cells is visible (arrows). Anterior is towards the left. Scale
bar: 10 μm.

Penetrance of the vulval lin-11::GFP expression at different
developmental stages in two transgenic lines: syIs80 (A-C) and
syIs53 (D,E). x-axis is the vulval cells, whereas the
y-axis is the percentage of animals expressing GFP (sample
size for each set is 50). (A) GFP penetrance is higher in P5.pp and P7.pa
cells compared with others. syIs53 animals do not reveal vulval
expression at this stage. (B,D) By Pn.pxx stage, P5.p and P7.p N,T cells
express high levels of GFP. The penetrance is complete in
syIs80 animals but lower (60-70%) in syIs53 animals. (C,E)
By Pn.pxxx stage, GFP expression is detected in all 2° lineage
cells.

By mid-L4 stage, the GFP fluorescence in syIs53 and
syIs80 strains begins to fade, and can not be seen by late-L4 stage.
However, in nIs96 animals fluorescence can be detected in young adult
animals. Expression is also detected in the uterine π lineage cells, VC
neurons and a subset of the head and tail neurons in a manner similar to that
reported earlier (Hobert et al.,
1998; Newman et al.,
1999). In addition, we observe expression in the B.pap and its
descendents in the developing male proctodeum.

Early expression of lin-11 in vulval cells determines the pattern of
invagination

The vulval expression of lin-11 suggests an earliest requirement
in VPC daughters (Pn.px cells). In wild-type animals, the vulC and vulD cells
of the 2° lineage invaginate during L4 stage
(Fig. 1). A high level of
lin-11 expression in their precursors at Pn.px and Pn.pxx stages
suggests that in wild-type animals, LIN-11 activity could specify the ability
of cells to invaginate, consistent with the vulval invagination defect
observed in lin-11 mutant animals. To determine whether ectopic
expression of lin-11 can alter vulval cells fates and therefore
invagination pattern, we generated transgenic animals carrying full-length
lin-11 genomic DNA under the control of the heat-shock promoter,
hsp16-41. Such animals (hs::lin-11) were heat shocked at
different stages (Pn.px, Pn.pxx and Pn.pxxx) and analyzed for the vulval
morphology phenotype. Although the heat shock given at the Pn.pxxx stage did
not cause a noticeable defect in vulval morphology, heat shocks at the other
two stages caused ectopic invagination
(Fig. 6). Specifically, the
vulA, vulB1 and vulB2 cell types that normally remain adhered to the epidermis
in wild type had invaginated (Fig.
6A,C;
compare with wild-type in Fig.
7A). The defect was qualitatively similar after the heat shock at
Pn.px or Pn.pxx stage, although the penetrance was higher at the Pn.px stage.
In most cases, only a subset of the P5.p and P7.p lineage cells showed ectopic
invagination (90%, n=19; Fig.
6A), although in one animal all vulval cells were completely
invaginated (Fig. 6C). This
phenotype suggests that ectopic expression of lin-11 in the
precursors of vulA, vulB1 and vulB2 interferes with their normal development,
and possibly alters their cell fates. This hypothesis was further supported by
our observation that in some cases (two out of six) ectopically invaginated
cells showed expression of the egl-17::GFP, a marker for wild-type
vulC and vulD cell fates (see Fig.
6B for ectopic expression in P7.p lineage vulA; Fig.
3A,B
shows wild-type pattern). By contrast, no such defect was observed in control
heat shock experiment. We conclude that during Pn.px and Pn.pxx stages
lin-11 expression in the precursors of vulC and vulD promotes a
wild-type vulval invagination.

Effect of the heat shock induced lin-11 expression. (A) Pn.pxx
stage heat pulse causes ectopic invagination in some of the P5.p and P7.p
lineage cells. (B) The same animal as in A. The ectopic expression of
egl-17::GFP can be seen in the presumptive vulA of the P7.p lineage.
(C) In this animal (heat pulsed during Pn.px stage), all vulval cells have
invaginated. The hs::lin-11 transgenic strains are syEx530
(A,B) and syEx500 (C). utse, uterine seam cell. In all images,
anterior is towards the left. Scale bar: 8 μm.

Vulval morphogenesis defects caused by lin-11 RNAi. (A,B)
Wild-type. (C-E) Early Pn.pxxx stage heat shocked hs-dslin-11i
animals. (A) During mid-L4 stage, the vulD nuclei are located in the same
plane of focus (arrows). (B) Same animal as in A when viewed at different
focal plane. Thick arrows mark the 1° lineage cell nuclei, vulE and
vulF–all seen together. (C) A hs-dslin-11i animal having
defects in the positioning of vulval nuclei. In this focal plane, only the
P5.p lineage presumptive vulD nucleus is visible. (D) The same animal as in C
when viewed at different focal plane. The presumptive vulD nucleus of the P7.p
lineage (thin arrow) is seen along with the vulF nuclei (thick arrows). vulE
pair is not visible in this plane. (E) A weak Pvul phenotype seen in some
hs-dslin-11i adults. Anterior is towards the left. Scale bars: in A,
8 μm for A-D; in E, 10 μm.

In addition to the invagination defects, we observed a weak multivulva
(Muv) phenotype from the heat shocks given during the early Pn.px stage (16%,
n=69; average VPC induction 3.2). Two major signaling pathways that
function during vulval induction are the EGF-receptor and LIN-12/Notch
mediated pathways (reviewed by Greenwald,
1997; Wang and Sternberg,
2001). We tested the involvement of EGF-receptor signaling using a
hypomorphic lin-3 allele, n378, and observed complete
suppression of the Muv phenotype. In addition, hs::lin-11 does not
suppress the vulval induction defect of n378 (VPC induction 0.8,
n=36 compared with the control n378 heat-shocked animals
0.7, n=35). This epistasis of n378 over hs::lin-11
suggests that the effect of lin-11 overexpression is likely to be
upstream of lin-3, and possibly at the level of lin-3
transcription. We are not convinced that such an effect of lin-11 is
physiologically relevant because none of the known alleles of lin-11
exhibit defects in the extent of vulval induction, and lin-11::GFP
transgenic animals (nIs96, syIs80 and syIs53) do not reveal
GFP fluorescence in the anchor cell (AC) during L2/L3 stages when AC is
required for vulval induction. It is more likely that the overexpression of
lin-11 mimics the effect of some other homeodomain or LIM homeodomain
protein.

Our experiments so far have defined the function of lin-11 during
the Pn.px and Pn.pxx stages in establishing the correct pattern of vulval
invagination. lin-11 continues to be expressed at high levels in
Pn.pxxx cells and thus might be required for vulval differentiation. To test
this hypothesis, we used a conditional RNAi approach to inactivate
lin-11 gene function. We generated transgenic hs-dslin-11i
animals (carrying lin-11 cDNA in sense and antisense orientations
under control of the hsp16-41 heat-shock promoter) that can be heat
shocked at any desired developmental stage to induce the formation of
double-stranded RNA. As a control, we heat shocked hs-dslin-11i
animals during early L3 stage (Pn.p cells in
Fig. 1) and compared the vulval
morphology and egg-laying phenotypes with the lin-11 loss of function
alleles. Forty percent (n=8) of the heat-shocked animals showed an
egg-laying defective (Egl) phenotype and a weak vulval invagination defect
similar to the lin-11(n566) allele. One of them also exhibited the AC
migration defect, a phenotype that contributes to the Egl defect in
lin-11 mutant animals (Newman et
al., 1999). In control heat-shocked animals (no
hs-dslin-11i), no such defect was observed (n=20). These
results confirm that the RNAi phenotypes of hs-dslin-11i animals are
due to the reduction in the wild-type lin-11 gene function. Next, we
examined the effect of the lin-11 RNAi on vulval morphology by heat
shocking hs-dslin-11i animals during early Pn.pxxx stage (early-L4).
In wild-type animals during the mid-L4 stage, vulval nuclei occupy
stereotypical positions, such that vulD nuclei of the P5.p and P7.p lineage
are located in the same plane of focus
(Fig. 7A). Likewise, vulE and
vulF nuclei are seen in one focal plane, different from that occupied by vulD
nuclei (Fig. 7B). By contrast,
the heat-shocked hs-dslin-11i animals showed significant defects in
the vulval morphology with misplaced vulval nuclei (30%, n=16; Fig.
7C,D).
Specifically, we found that vulC and vulD nuclei were located in wrong focal
planes (vulD is shown in Fig.
7C,D).
Two out of five defective animals also showed abnormal positioning of the
nuclei of 1° lineage cells (see Fig.
7D for vulF position, vulE nuclei are not seen in this plane).
Overall, vulval invagination was narrower along the anteroposterior axis
compared with the wild type. This abnormal morphology was correlated with a
protruding vulva phenotype at the adult stage
(Fig. 7E). However, such
animals were able to lay eggs normally.

We also examined egl-17::GFP expression in lin-11 RNAi
animals during the mid-L4 stage (∼4 hours after the heat shock treatment).
Although the overall GFP pattern was qualitatively wild type (n=9;
see Fig.
3A,B
for the wild-type egl-17::GFP pattern), two worms did show moderate
reduction in the GFP fluorescence in vulC and vulD. These results reveal a
novel function of lin-11 in vulval morphogenesis, distinct from its
early role in specifying the invagination pattern.

The LIM binding protein LDB-1 plays a role in vulval patterning

The LIM homeodomain proteins contain a pair of LIM domains that interact
with co-factors and modulate protein activity
(Dawid et al., 1998;
Bach, 2000;
Hobert and Westphal, 2000).
Among the co-factors are the LIM-binding proteins represented by
NLI/Ldb1/CLIM2, which display highly specific interactions with the LIM
domains. The C. elegans LIM-binding protein LDB-1 has been shown to
interact with two LIM homeodomain proteins CEH-14 and MEC-3
(Cassata et al., 2000). As it
is the only known LIM-binding protein in C. elegans, it is likely
that LDB-1 regulates the activities of other LIM homeodomain proteins,
including LIN-11.

To investigate the role of ldb-1 during vulval development, we
examined its expression using a ldb-1::GFP construct.
ldb-1::GFP expression is first detected in embryos towards the end of
gastrulation (prior to the comma stage) and is seen in the vulval cells among
other expression (Cassata et al.,
2000) (not shown). In vulval cells ldb-1::GFP expression
is observed in both the 1° as well as 2° lineage cells
(Fig. 8). Expression during the
Pn.px stage was rarely observed (<5%, n=28;
Fig. 8I). During Pn.pxx stage,
a weak and low penetrant GFP could be detected in 1° and 2°
lineage cells (16%, n=25; Fig.
8A,B,I).
However, by the Pn.pxxx stage strong and highly penetrant ldb-1::GFP
expression could be detected in all vulval progeny (Fig.
8C-F,I).
Expression was also observed during early adult stage
(Fig. 8G-I).

We examined the effect of decreasing ldb-1 activity using RNAi.
ldb-1 RNAi animals showed uncoordinated movement and failed to
respond to touch, a phenotype that has been previously described (Cassatta et
al., 2000) (not shown). We also observed defects in vulval morphology and
gonad arms at significant frequencies (vulval defect: 26%, n=101, see
Fig. 9; gonad defect: 40%,
n=18). Vulval phenotypes included abnormal placement of the 1°
and 2° lineage cells (Fig.
9A,C,E).
Occasionally, animals displayed a protruding vulva phenotype and were Egl. We
did not observe a vulval invagination defect in any of the RNAi animals. We
also examined the effect of ldb-1 RNAi on vulval markers,
egl-17::GFP, zmp-1::GFP, cdh-3::GFP and ceh-2::GFP. In all
but the case of zmp-1::GFP, GFP fluorescence was altered
(Fig. 9, compare with wild-type
patterns in Fig. 3).
egl-17::GFP expression was frequently absent in the presumptive vulC
(Fig.
9A,B).
The cdh-3::GFP showed reduced or no expression (Fig.
9C,D
shows only vulD expression). ceh-2::GFP expression in the presumptive
vulB1/B2 cells was variable and weak, whereas presumptive vulC often showed no
expression (Fig.
9E,F).
In control RNAi animals, no such defects were observed. Thus, LDB-1 plays a
role in vulval morphogenesis. In Drosophila, the LIM homeodomain
protein Apterous and its LIM-binding partner Chip form a regulatory feedback
network to modulate the expression of each other
(Milan and Cohen, 2000). We
examined the possibility of such feedback regulation between LDB-1 and LIN-11,
but found no change in the expression of lin-11::GFP in
ldb-1 RNAi animals.

LDB-1 and LIN-11 interact in yeast two-hybrid assay

We examined physical interaction between LIN-11 and LDB-1 using a
two-hybrid interaction assay (Fields and
Song, 1989). For this, we designed two separate expression
constructs. One construct expresses LIM domains of LIN-11 as a fusion protein
with the GAL4 DNA-binding domain, whereas the other expresses LDB-1
LIM-interacting domain (LID) fused to the GAL4 activation domain (see
Materials and Methods). Fig.
10B shows that fusion proteins involving LIN-11 LIM domains and
LDB-1 LID interact with each other thereby leading to the growth of yeast
cells on plates lacking histidine, leucine and tryptophan. Neither one alone
(Fig.
10B-2,B-3)
is sufficient for HIS3 expression. In a control experiment
(Fig. 10A), all transformants
grew on plates lacking leucine and tryptophan. These results demonstrate a
physical interaction between LIN-11 and LDB-1.

Yeast two-hybrid interaction test of LIN-11 and LDB-1. Yeast cells were
transformed with plasmids pGBKT7 and pGADT7 (1), pGBKT7-lin-11 and pGADT7 (2),
pGBKT7 and pGADT7-ldb-1 (3), pVA3 and pTD1 (4), and pGBKT7-lin-11 and
pGADT7-ldb-1 (5). In a transformation control (A), all cells can be seen
growing on plate that lacks leucine and tryptophan (SD/–Leu/–Trp).
However, only positive control (pVA3 and pTD1) and test (pGBKT7-lin-11 and
pGADT7-ldb-1) can promote HIS3 expression (B), leading to growth on
plate that lacks histidine, leucine and tryptophan
(SD/–His/–Leu/–Trp).

In wild-type C. elegans during the L4 stage, vulval cells initiate
the process of invagination (Fig.
1) and specific cell fusion to give rise to the adult structure.
The cell fusion events are ordered and occur only between the homologous cell
types, e.g. P5.p lineage vulA fuses only with the P7.p lineage vulA
(Sharma-Kishore et al., 1999).
By contrast, lin-11 mutant vulval cells exhibit defects in cell
fusion events. Often all 2° lineage vulval cells fuse together, suggesting
that they have acquired a common fate. However, this fate is distinct from any
of the wild-type cell fates as none of the examined markers (egl-17::GFP,
zmp-1::GFP, cdh-3::GFP and ceh-2::GFP) is expressed in
lin-11 mutant vulval cells. Using cell ablation experiments, we have
shown that defects in cell fusion events and marker gene expression in
lin-11 mutant animals result from cell-autonomous requirements of
lin-11. The phenomenon of a cell fusion defect is similar to that
observed for ray fusion in C. elegans mutants affecting male tail
development (Baird et al.,
1991; Chow and Emmons,
1994).

Analysis of the ajm-1::GFP expression in 1° vulval cells in
lin-11 animals has revealed defects in the morphology of vulE and
vulF rings, consistent with the abnormal patterns of egl-17::GFP,
cdh-3::GFP and zmp-1::GFP expression (see
Fig. 3). Hence, the 1°
lineage cells are not likely to form a functional vulval opening. These
results are consistent with our previous findings on tissue-specific
regulation of lin-11, where we used vulval- and uterine-specific
regulatory elements of lin-11 to demonstrate that wild-type
egg-laying requires lin-11 function in both the vulva and the uterineπ
lineage cells (Gupta and Sternberg,
2002).

The vulval cell fusion defects in lin-11 mutant animals could
arise because lin-11 directly regulates the process of cell fusion or
as a consequence of abnormal differentiation. Two sets of results support the
latter possibility. First, lin-11 expression in vulval cells is
detected beginning at the Pn.px stage (see
Fig. 4). Second, induction of
lin-11 RNAi (using hs-dslin-11i) during mid-L4 stage causes
no significant effect on the formation of vulval rings. Thus, during terminal
differentiation, lin-11 mutant vulval cells might fail to express
cell-type specific genes, leading to the defects in fate specification. The
abnormal cell fusion is the consequence of cells failing to acquire their
unique identities. Such a role of lin-11 in the vulva is similar to
that of C. elegans LIM homeobox gene, mec-3, in touch
receptor neurons. In mec-3 mutant animals, the presumptive touch
neurons are generated but fail to acquire the correct identity
(Way and Chalfie, 1988).

Temporal expression of lin-11 promotes distinct vulval cell
fates

LIM homeobox genes have been shown to express in highly restricted spatial
and temporal manner (Bach,
2000; Hobert and Westphal,
2000). Wing development in Drosophila requires dynamic
expression of the LIM homeobox gene apterous. The level and domain of
apterous expression are highly regulated and help define the
dorsoventral boundary leading to wing growth and patterning
(Diaz-Benjumea and Cohen,
1993; Milan and Cohen,
2000).

The dynamic expression of lin-11 in vulval cells can be classified
into two distinct patterns: an initial polarized expression (during Pn.px and
Pn.pxx stages) where only a subset of the cells express lin-11, and a
broad pattern of expression during terminal differentiation (Pn.pxxx cells)
where all 2° lineage cells express lin-11. We hypothesized that
these two different patterns of lin-11 expression may have different
functions and tested the hypothesis experimentally. First, a
hs::lin-11 system was used to express lin-11 ectopically in
all vulval cells during Pn.px and Pn.pxx stages. This led to defects in vulval
invagination caused by the failure of presumptive vulA, vulB1 and vulB2 to
remain adhered to the epidermis (Fig.
6; wild-type pattern in Fig.
1). Second, using a RNAi approach, we inhibited lin-11
function during early Pn.pxxx stage when lin-11 is expressed in all
2° lineage cells. The lin-11 RNAi animals showed defects in
vulval morphology and vulval nuclei failed to occupy stereotypic positions.
This phenotype is likely to result from a differentiation defect in vulval
progeny.

The two distinct requirements of lin-11 in vulval cells are likely
to be mediated by different target genes. The vulval invagination defect in
lin-11 animals suggests that one potential target of lin-11
could be the genes that regulate epithelial morphogenesis. Our reporter gene
expression studies have identified a cadherin family member, cdh-3,
that functions downstream of lin-11. In lin-11 mutant vulval
cells cdh-3::GFP expression in the presumptive vulC and vulD is
abolished (Fig. 3;
Table 1). Cadherins are known
to regulate epithelial morphogenesis by mediating adhesions between cell-cell
and cell-extracellular matrix (Gumbiner,
1996). However, the function of cdh-3 in vulval
development is not essential, perhaps owing to redundancy.

The early expression of lin-11 is polarized and confers identity
on cells to give rise to progeny (vulC and vulD) that invaginate during L4
stage (see Fig. 1). This
conclusion is also supported by the roles of lin-17 (a
frizzled family member) (Sawa et
al., 1996) and lin-11 during vulval development
(Gupta and Sternberg, 2002).
In lin-17 mutant animals, lin-11 expression in P7.p lineage
cells is often reversed, i.e. LL lineage cells begin to express
lin-11 instead of the wild-type NT lineage cells. This reversal in
the polarity of lin-11 expression correlates with the opposite
orientation of invagination of the P7.p lineage cells. A similar role for
lin-11 has also been demonstrated in the specification of the ASG and
AWA neurons (Sarafi-Reinach et al.,
2001). Although during embryonic stages both neurons express
lin-11, expression in AWA is lost by the L1 larval stage and persists
only in the ASG neuron. This later stage expression of lin-11 in ASG
is necessary for its wild-type development. If lin-11 is ectopically
expressed in AWA during post-L1 larval stages, the AWA adopts partial ASG-like
features. Similar functions of other LIM homeobox genes in determining
polarity or asymmetric cell fates have also been demonstrated. C. elegans
lim-6, another LIM homeobox gene, generates functional differences in the
chemosensory behavior between a pair of neurons ASEL/R
(Pierce-Shimomura et al.,
2001). In mouse embryonic axis formation, the role of
lim1 in anterior-posterior polarity is also suggestive of such a
biological function (Perea-Gomez et al.,
1999). In this case, lim1 expression in the anterior
region cells of the visceral endoderm confers anterior identity and makes them
different from the posterior cells. Thus, the role of the LIM homeobox genes
in generating cellular asymmetry appears to be a conserved biological
function.

Functional specificity of LIN-11 during vulval development

How does lin-11 play different roles at different stages of vulval
development? One possibility could be that LIN-11 interacts with stage- and
cell-type specific factors to bring about the different outcomes. The LIM
domains of the LIM homeodomain proteins are known to serve as protein-protein
interacting interface that promote the formation of multimeric complexes and
influence DNA-binding affinity of the homeodomain
(Dawid et al., 1998). Many
studies have revealed the presence of LIM domain-binding proteins
(Dawid et al., 1998;
Bach, 2000;
Hobert and Westphal, 2000).
Although a majority of them belong to the NLI/Ldb1/CLIM2 family, others such
as POU homeodomain factor Pit1 (Bach et
al., 1995), WD40 repeat containing factor SLB
(Howard and Maurer, 2000) and
bHLH factor E47 (German et al.,
1992) have also been identified.

The C. elegans LIM-binding protein LDB-1 was previously shown to
be required for the wild-type functioning of several neurons
(Cassata et al., 2000). We find
that ldb-1 is expressed in both the 1° as well as 2° lineage
vulval cells, a pattern that overlaps with lin-11 expression.
Analysis of the ldb-1 vulval expression has revealed some differences
from lin-11. During Pn.pxx stage, lin-11::GFP shows
alternating low and high pattern of expression in P5.p and P7.p lineage cells
(LLHH-HHLL, respectively, from anterior to posterior; L, low; H, high).
ldb-1::GFP, however, shows no such pattern and is detected at uniform
level in all 1° and 2° lineage cells. In addition, ldb-1::GFP
continues to be expressed at high levels in 1° vulval progeny during L4
and young adult stages, whereas lin-11::GFP expression in 1°
lineage cells is significantly weaker compared with the 2° lineage cells.
Hence, LDB-1 may regulate only a subset of the LIN-11 functions. Consistent
with this, ldb-1 RNAi animals did not show defects in vulval
invagination but in vulval morphology (Figs
7,
9). However, it is possible
that ldb-1 RNAi effect is weak because of the partial elimination of
gene activity. Our findings that LIN-11 and LDB-1 physically interact support
the hypothesis that LIN-11 and LDB-1 function together to regulate vulval
differentiation. Furthermore, these results suggest that lin-11 may
use other mechanisms during earlier stages of vulval development.

Acknowledgments

We thank S. Cameron, D. Sherwood and T. Inoue for integrated strains.
Robert Oania provided assistance in two-hybrid experiments. Some of the
strains were obtained from Caenorhabditis Genetics Center. We also
thank D. Sherwood, T. Inoue and anonymous reviewers for comments on the
manuscript. This work was supported by funds from the HHMI and USPHS grant
HD23690 to P.W.S. B.P.G. was supported by a long-term fellowship from HFSP and
as an associate of the HHMI. M.W. was an AMGEN fellow. P.W.S. is an
investigator of the HHMI.

Other journals from The Company of Biologists

This poster and accompanying article from Niki Anthoney, Istvan Foldi and Alicia Hidalgo highlights the diverse, context-dependent roles that Toll/TLRs play beyond innate immunity in determining cell fate and differentiation, including during competition for light in plants and in neurotrophism and plasticity in the CNS.

Nicholas E. Baker and Nadean L. Brown review how gene duplication and divergence are interwoven with neuronal complexity in Drosophila and vertebrates, highlighting atonal as a platform for understanding proneural gene structure and regulation.

We are currently seeking proposals for four Workshops to be held in 2020. Do you have an idea for a Workshop? Please let us know and you could be one of our 2020 Workshop organisers. You focus on the science, we focus on the logistics. We are particularly keen to receive proposals from postdocs. Deadline date for applications is 25 May 2018.

Development is a proud sponsor of the upcoming Santa Cruz Developmental Biology Meeting, which takes place 11-15 August 2018 at the University of California, Santa Cruz . Registration for this meeting is now open!

Meet the preLighters! In the latest interview with our preLights community, the preLights team caught up with James Gagnon, Assistant Professor at the University of Utah, to talk about his research, how science can be made more open, his enthusiasm for the preLights project and the fun sides of being a junior PI.

To investigate which signalling pathways are regulated by nitric oxide during mouth development in Branchiostoma lanceolatum (amphioxus), Filomena Caccavale used a Travelling Fellowship from Development to visit The Oceanographic Observatory in Banyuls-sur-Mer, France, an area with a thriving natural amphioxus population. Read more on her story here.

Where could your research take you? Join Filomena and apply for the next round of Travelling Fellowships from Development by 25 May 2018.